This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. Inhibition of the mannose trimming enzyme human Golgi [unreadable]-mannosidase II (HGMII), which acts late in the N-glycan processing pathway, provides a route to blocking the oncogene-induced changes in cell surface oligosaccharide structures. HGMII selectively cleaves [unreadable](1-3) and [unreadable](1-6) mannosyl residues present in its natural substrate GlcNAcMan5GlcNAc2. It is a retaining glycosylhydrolase, which employs a two-stage mechanism involving two carboxylic acids positioned within the active site to act in concert: one as a catalytic nucleophile and the other as a general acid/base catalyst. Protonation of the exocyclic glycosyl oxygen of a substrate molecule leads to bond-breaking and simultaneous attack of the catalytic nucleophile to form a glycosyl enzyme intermediate. Subsequent hydrolysis of the covalent intermediate by a nucleophilic water molecule gives an [unreadable]-mannose product with overall retention of configuration. In order to probe the substrate requirements of HGMII, we have synthesized a number of oligosaccharides, which were used in co-crystallization studies with Drosophilia Golgi [unreadable]-mannosidase II (dGMII). A co-crystal structure GlcNAcMan5 with a mutant enzyme in which the catalytic nucleophilic acid has been replaced by Ala, uncovered the molecular interactions of the enzyme with the [unreadable](1,2)-linked GlcNAc moiety of the natural substrate. The structure of the co-complex bound to an extended cleft leading from the glycone binding site to the accessory GlcNAc binding site is consistent with the >100-fold preference of dGMII for cleavage of substrates containing a non- reducing [unreadable](1,2)GlcNAc residue. By contrast, the absence of an equivalent GlcNAc binding site in lysosomal and kinetic analysis indicating that the latter enzyme prefers oligomannose substrates without non-reducing terminal GlcNAc modifications, suggests that selective inhibitors for GMII could exploit the additional binding specificity of the GlcNAc binding site.